Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis

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Abstract

The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.
Original languageEnglish
JournalMethods in Molecular Biology
Volume873
Pages (from-to)297-305
Number of pages9
ISSN1064-3745
DOIs
Publication statusPublished - 2012

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Isotope Labeling
Amino Acids
Proteins
Membranes
Human Embryonic Stem Cells

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title = "Stable isotope labelling with amino acids in cell culture for human embryonic stem cell proteomic analysis",
abstract = "The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.",
author = "Linda Harkness and Prokhorova, {Tatyana A} and Moustapha Kassem and Blagoy Blagoev",
year = "2012",
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AU - Harkness, Linda

AU - Prokhorova, Tatyana A

AU - Kassem, Moustapha

AU - Blagoev, Blagoy

PY - 2012

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AB - The identification and quantitative measurements of proteins in human embryonic stem cells (hESC) is a fast growing interdisciplinary area with an enormous impact on understanding the biology of hESC and the mechanism controlling self-renewal and differentiation. Using a quantitative mass spectroscopic method of stable isotope labelling with amino acids during cell culture (SILAC), we are able to analyse differential expression of proteins from different cellular compartments and to identify intracellular signalling pathways involved in self-renewal and differentiation. In this chapter, we provide a detailed method for creating SILAC media suitable for use in hESC experiments, additionally we describe methods for the isolation of membrane fractions and cytosolic and nuclear/membrane fractions.

U2 - 10.1007/978-1-61779-794-1_20

DO - 10.1007/978-1-61779-794-1_20

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JF - Methods in Molecular Biology

SN - 1064-3745

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